RESEARCH ARTICLE Open Access

Cells of renin lineage express hypoxiainducible factor 2α following experimentalureteral obstructionAnia Stefanska1, Diana Eng1, Natalya Kaverina1, Jeffrey W. Pippin1, Kenneth W. Gross2, Jeremy S. Duffield3

and Stuart J. Shankland1*

Abstract

Background: Recent studies indicate that mural cells of the preglomerular vessels, known as cells of renin lineage(CoRL), contribute to repair and regeneration of injured kidney glomeruli. However, their potential roles intubulointerstitial disease are less understood. The aim of this study was to better understand CoRL numberand distribution following UUO so that future mechanistic studies could be undertaken.

Methods: We mapped the fate of CoRL in adult Ren1cCreER x Rs-tdTomato-R reporter mice that underwent UUO.Kidney biopsies from sham and UUO-subjected mice on days 3, 7, and 14 were evaluated by immunohistochemistry.

Results: In sham animals, CoRL were restricted to juxtaglomerular location. At day 7 following UUO, CoRL increasedtwo-fold, were perivascular in location, and co-expressed pericyte markers (PDGFßR, NG2), but did not express renin.At day 14 post UUO, labeled CoRL detached from vessels and were present in the interstitium, in areas of fibrosis,where they now expressed the myofibroblast marker alpha-smooth muscle actin. The increase in CoRL waslikely due to proliferation as marked by BrdU labeling, and migration from the cortex. Following UUO startingfrom day 3, active hypoxia inducible factor-2α was detected in nuclei in labeled CoRL, in the cortex, but notthose cells found in medulla.

Conclusions: We have demonstrated that arteriolar CoRL are potential kidney progenitors that may contributeto the initial vascular regeneration. However, in chronic kidney injury (≥14 days post UUO), perivascular CoRLtransition to myofibroblast-like cells.

Keywords: HIF-2α, UUO, Microvascular rarefaction, Pericytes, progenitor, Tubulointerstitial, PDGFßR, Fibrosis,Hypoxia

BackgroundUnilateral ureteral obstruction (UUO), a commonlyused experimental model of chronic kidney injury ischaracterized by tubular atrophy, inflammation andinterstitial fibrosis [1, 2]. In UUO, the initiating damageis increased ureteral pressure transmitted retrograde tothe kidney that causes secondary renal vasoconstrictionand resultant reduced glomerular blood flow [3]. If thepressure is not relived, the renal vascular resistance re-mains increased, causing ischemia (reviewed in [4]).

The resultant tubulointerstitial fibrosis in UUO ismulti-factorial, including interstitial macrophages pro-ducing pro-inflammatory cytokines, tubular cells undergo-ing apoptosis, and resident renal cells transitioning tocollagen-producing cells [1]. The origin of the collagenproducing cells has been attributed to the perivascular cellpopulation, namely pericytes and perivascular fibroblasts.Following UUO, perivascular cells become activated, anddetach from the underlying vessels. The consequences ofpericyte detachment include that endothelial cells aredeprived of survival factors [5], vascular tubes becomeunstable and more permeable leading to microvascularrarefaction [6], and migrating perivascular cells can de-differentiate into myofibroblasts, thereby becoming asource of collagen [7, 8].

* Correspondence: stuartjs@uw.edu1Department of Medicine, Division of Nephrology, University of Washington,Seattle, WA 98104, USAFull list of author information is available at the end of the article

© 2016 Stefanska et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, andreproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link tothe Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Stefanska et al. BMC Nephrology (2016) 17:5 DOI 10.1186/s12882-015-0216-0

Less is understood the reparative, or attempted repara-tive processes, if any, in UUO. Cells of renin lineage(CoRL) refer to all possible cellular derivatives originatingfrom renin expressing cells at time captured by reporting.Fate tracking studies showed that in development, CoRLgive rise to juxtaglomerular (JG) cells producing renin andto non-renin-producing cells such as smooth muscle cells,mesangial cells, tubular cells and extrarenal cells [9]. Re-cently, the lineage relationship between Ren+ and Foxd1+stromal cells was clarified when it was revealed that allmural cells, including renin cells, are derived from Foxd1stromal cells [10]. Studying CoRL (Ren+ progenitors) pro-vides an opportunity to characterize in depth a subpopula-tion of stromal cells. It is important because we needbetter understanding of different subpopulation of stromalcells to design specific therapeutic interventions [11].Pericytes have gained recognition in the kidney for their

role in pathogenesis of fibrosis [7, 8]. Interestingly, CoRLhave been acclaimed as a candidate progenitor of thekidney. Several studies showed that CoRL can regeneratemesangial cells [12, 13], podocytes [14], parietal epithelialcells [14], and erythropoietin-producing cells [15]. Themost recent study from our lab demonstrates that inglomerular injury and remnant kidney models, CoRL mi-grate to the interstitium and regenerate into pericytes [16].The current study was designed to further explore the roleof CoRL in progressive tubulointerstitial injury, the experi-mental model of unilateral ureteral obstruction.

MethodsAnimalsTo study the fate of renin lineage cells in chronic kidneydisease, we used RenCreER (Ren1cCreERxRs-tdTomato)transgenic mice on a mixed C57 BL10/C3H background[16]. In RenCreER mice, cells of renin lineage are onlylabeled permanently in inducible manner with tdTomatored protein within temporal windows defined by theadministration of tamoxifen. 8–9 week-old mice weregiven Tamoxifen (100 mg/kg) by IP injection for 6 days onalternate days, as we have previously reported [16, 17]. Wewaited at least 7 weeks between giving tamoxifen andinducing the disease model, to allow significant washoutof tamoxifen to exclude possibility of recombination inother cell types. Animal protocols were approved by theUniversity of Washington Institutional Animal Care andUse Committee (2968-04).

Experimental model of kidney fibrosisUnilateral ureteral obstruction (UUO) was performed inadult female mice, as previously described [7, 18, 19].Briefly, mice were anesthetized with isofluorane (1 %,inhaled). UUO was induced by left ureteral ligation usinga 4-0 silk tie suture at two points. Sham operated miceunderwent the same procedure except that left ureter was

only exposed by flank incision, served as controls (n = 6).Kidneys were harvested on d3 (n = 6), d7 (n = 17), and d14(n = 6).

Assessment of kidney fibrosisHistological analysis of fibrosis was performed on fixedrenal tissue, embedded in paraffin, and sectioned at athickness of 4 μm. Connective tissue deposition wasexamined with Picrosirius Red Stain Kit (Polysciences, Inc,Warrington, PA, USA) and collagen I (1:100, Millipore,Billerica, MA, USA), staining, as we have previouslydescribed [20–22]. Additionally, co-staining of CoRL and amyofibroblast marker alpha smooth muscle actin (αSMA,1:10.000; Sigma, Saint Louis, MI, USA) were performed todetermine whether CoRL become myofibroblasts in kidneyfibrosis.

Detection of tdTomato reporterIn order to visualize tdTomato reporter labeling used tolabel CoRL in RenCreER mice, kidneys were fixed in10 % buffered formalin, embedded in paraffin, andsectioned at a thickness of 4 μm. Kidney sections under-went deparaffinization, heat-mediated antigen retrieval incitrate buffer pH 6.0, and blocking unspecific background(Accurate, San Jose, CA, USA). Immunofluorescent stain-ing for the tdTomato reporter was performed with Dye-Light 594-conjugated RFP (Red Fluorescent Protein) rabbitantibody (1:100, Rockland Immunochemicals for Research,Gilbertsville, PA, USA) at room temperature for 1.5 h.tdTomato fluorescent signal is discernable in non-paraffin-embedded tissue only.

Identification of cell proliferationBrdU (5-Bromo-2-deoxyridine) incorporation assay wasused to quantitate cell cycle entry and proliferation. 10 μlof BrdU (Amersham Cell Proliferation Labeling Reagent,GE Healthcare Life Sciences, Little Chalfont, UK) per grambody weight was administered via IP on alternate days fol-lowing UUO. Double immunostaining was performed forBrdU and tdTomato. Antibody paraffin embedded tissuewas prepared and blocked as described above. Avidin/bio-tin blocking (Vector Laboratories, Burlingame, CA, USA)was performed to block endogenous biotin and preventunspecific staining while using biotin-streptavidin labelingsystem. Tissue was incubated overnight at 4 °C with aprimary mouse anti-BrdU (1:200, Amersham, GE LifeSciences, Buckinghamshire, UK). This was followed with abiotinylated goat anti-mouse antibody (1:500, JacksonImmunoresearch Laboratories, Inc, West Grove, PA,USA) incubated at room temperature for 1 h. Thesignal was amplified by incubation with streptavidin-conjugated with Alexa Fluor 488 (1:100; Invitrogen,Grand Island, NY, USA) for 45 min. Negative control

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staining was performed by omitting primary antibodystaining.

Assessment of vascular changesEndothelial marker staining CD31 (PECAM-1) wasperformed to examine changes in microvascular dens-ity, and to demonstrate any perivascular location ofCoRL and pericytes. Rat anti-mouse CD31 (1:100;Dianova, Hamburg, Germany) was incubated over-night at 4 °C following incubation with secondaryanti-rat antibody conjugated with Alexa Fluor 647(1:100 Invitrogen).Pericytes were identified by the expression of NG2 and

PDGFRß. To identify pericytes derived from CoRL, tripleimunostaining was performed on frozen tissue sections asfollows. Rabbit anti-NG2 antibody (1:100; Millipore,Billerica, MA, USA) was incubated overnight at 4 °C,followed by biotinylated anti-rabbit antibody (1:500; Vec-tor) incubation at room temperature for 1 h, and strepta-vidin conjugated with Alexa Fluor 647 (1:100; Invitrogen,Grand Island, NY, USA) for 45 min. To prevent non-specific staining for the primary antibodies from the samespecies pre-incubation with anti-rabbit IgG Fab (1:25;Jackson ImmunoResearch Laboratories, West Grove, PA,USA) was followed by rabbit IgG Fab incubation (1:25;Jackson ImmunoResearch Laboratories). Rabbit anti-PDGFRß antibody (1:100; Abcam, Cambridge, MA, USA)was incubated with tissue sections overnight at 4 °C.Secondary donkey anti-rabbit antibody conjugated withAlexa Fluor 488 (1:100, Invitrogen) was incubated for 1 hat room temperature. Finally, anti-tdTomato antibody wasapplied.To examine hypoxia-activated locations in UUO, HIF-

2α (hypoxia inducible factor-2 α) staining was performedtogether with tdTomato reporter staining on frozen tis-sue sections. Rabbit anti-HIF-2α antibody (1:200; NovusBiological, Littleton, CO, USA) was incubated overnightat 4 °C, followed by biotinylated anti-rabbit antibody(1:500; Vector) incubation at room temperature for 1 h,and streptavidin conjugated with Alexa Fluor 647 (1:100;Invitrogen) for 45 min. Positive staining was assessedbased on nuclear HIF-2α localization confirmed byDAPI staining.

Image analysis and statistical analysisReporter positive-, BrdU- stainings were quantified on20 images of kidney cortex/medulla using 200x totalmagnification. Fluorescent imaging was performed usingEVOS®FL Cell Imaging System (Life Technologies). One-way ANOVA with Bonferroni post hoc test was used tocompare groups with P ≤ 0.05 as a criterion for statisticalsignificance. Data were presented as means ± SEM. Alldata were analyzed in GraphPad Prism 5.0 (GraphPadSoftware, La Jolla, CA, USA).

ResultsUUO results in microvascular rarefaction and increasedcollagen stainingAdditional file 1: Figure S1A shows the expected classicalhydronephrosis in the obstructed kidney as reported byothers [1, 4], with compensatory growth in the non-obstructed kidney. Prolonged ureteral obstruction leads torenal parenchymal damage and as a consequence loss ofkidney mass. Following UUO, there was a decrease of theligated/non-ligated kidney weight ratio d7 (1.00 ± 0.04 vs.0.79 ± 0.3, p < 0.01 vs. sham kidney) and d14 (1.00 ± 0.04vs. 0.64 ± 0.07, p < 0.001 vs. sham kidney) (Additional file 1:Figure S1B).Loss of microvessels affects oxygen supply and toxin

removal [23]. In chronic kidney disease, microvascularrarefaction contributes to tubulo-interstitial scarring andis a potential therapeutic target [19, 24]. Following UUO,there was a progressive decline in endothelial CD31staining in the cortex and medulla on d7 and d14 com-pared to the sham kidney (Additional file 1: Figure S1C).Picrosirius Red staining labels collagen I and III fibers. Asexpected, interstitial collagen levels were very low in shamkidneys (Additional file 1: Figure S1D). Following obstruc-tion, there was a progressive increase of picrosirius redstaining on days 3, 7, and 14 (Additional file 1: FigureS1D). The percentage area of Picrosirius Red stainingincreased from 3.05 ± 1.02 % in sham kidney to 5.61 ± 0.64on day 3 (P > 0.05), through 9.86 ± 1.48 on day 7 and wasthe highest on day 14 (14.03 ± 2.8, p < 0.01 vs. sham, andp < 0.05 vs. day 3) (Additional file 1: Figure S1E). Takentogether in this mouse strain, the decrease in endothelialcell staining and increase in fibrosis are consistent withclassic interstitial changes in UUO.

Interstitial CoRL number increases in UUOCortical changesDouble-staining was performed for RFP (to mark labeledCoRL), and CD31 (endothelial cell marker). Similar towhat we have previously reported [16], when CoRL re-porter mice are given tamoxifen to induce permanentCoRL reporting, and then undergo a sham procedure ascontrol, labeled CoRL (RFP positive, red color) in thecortex are typically localized and restricted to juxtaglo-merular (JG) compartment (Fig. 2a, arrowheads) andalong afferent arterioles (Fig. 1a, arrow). FollowingUUO, CoRL were also detected within the capillaryloops of an occasional glomerulus (Fig. 1b), and also inperitubular locations (Fig. 1c).In UUO there were no significant changes in the number

of total cortical CoRL (including JG, afferent arterioles,peritubular and glomerular CoRL) (Fig. 1d). There was aslight trend in the CoRL number increasing in the JG aftertwo weeks of kidney injury, however these changes werenot significant (Fig. 1e). Interestingly, at d3 post UUO,

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glomerular CoRL number increased compared to shamkidney (0.02 ± 0.02 vs. 0.15 ± 0.03, p < 0.01 vs. d3), and d14(0.04 ± 0.02 vs. d3, p < 0.05) (Fig. 1f). In contrast, peritubu-lar CoRL number increased significantly at d7 (0.23 ± 0.05vs. 0.04 ± 0.02, p < 0.05 vs. sham), (Fig. 1g). On d3 (0.13 ±0.05, p > 0.05) and d14 (0.19 ± 0.07, p > 0.05) followingUUO, there were trends towards an increase in peritubularCoRL number (Fig. 1g).

Medullary changesIn the medulla of sham kidneys, labeled CoRL weredetected in vasa recta (Fig. 1i). At d7, increased RFP la-beled CoRL were typically adherent to underlying vessels(Fig. 1j). Noteworthy was that at d14, the majority of

labeled CoRL were no longer adherent to underlying ves-sels, but rather were detected outside the vascular bed(Fig. 1k). Following UUO, there was a trend to an increasein the number of medullary CoRL at d3 (1.03 ± 0.19 vs.0.81 ± 0.17, p > 0.05 vs. sham). However, there was asignificant increase in medullary CoRL on d7 (1.62 ± 0.13,p < 0.01 vs. sham). The number of labeled CoRL was lowerat d14 post UUO (0.92 ± 0.21, p < 0.05 vs. d7), and was nodifferent from sham kidneys (P > 0.05) (Fig. 1h).These results show that the number of labeled CoRL in-

creases in peritubular locations in both cortex and medullaon d7. By d14, the majority of labeled (almost 100 %) CoRLhad detached from their vasculature, raising the possibilityof an ultimate myofibroblast fate.

Fig. 1 The number of cells of renin lineage (CoRL) increases beyond the JG location following UUO. CoRL were identified by RFP reporter staining (red),endothelial cells were labeled with CD31 staining (green). Nuclei are labeled with DAPI (blue). (a–g) CoRL in Cortex. (a) Representative images show that insham-operated mice, labeled CoRL (arrowheads) are predominantly localized to the juxta-glomerulus, with fewer detected along afferent arterioles (arrow).(b) At d7 post UUO, occasional labeled CoRL were detected in glomerular capillary loops. (c) Labeled CoRL were rarely detected in peritubular locations atday 14. Quantification: (d) Total cortical CoRL number was not statistically significant following UUO, (e) the same was found when CoRL were quantifiedexclusively in JG compartment. However, (f) there was a significant increase of intraglomerular CoRL at d3, and (g) peritubular CoRL at d7 andd14. (h–k) CoRL in Medulla. (h) Quantification - total CoRL number in the medulla was significantly increased on day 7 following UUO. (i) In the medullaof the sham kidney, CoRL were restricted to vasa recta. (j) Following UUO there was an increase of CoRL in vasa recta on day 7 (arrowheads).(k) 14 days after UUO, a subset of CoRL localized away from any vessels, being present in the interstitium

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CoRL proliferate and migrate following UUOHaving observed increased CoRL number in the medullafollowing UUO, we next asked whether this was due to cellproliferation by administering repeated BrdU pulses. As ex-pected, there was an increase of tubular cell BrdU stainingfollowing UUO, used as an internal positive control (Fig. 2b,c, open arrowheads). In the medulla, CoRL BrdU stainingwas only detected on UUO day 7 (Fig. 2b). Of the totalmedullary CoRL number, 3.78 ± 5.28 % stained for BrdU.Several proliferating medullary CoRL exhibited a markedshape change that gave the appearance of a migratoryphenotype (Fig. 2c, arrowhead). Moreover, using CD31staining to highlight endothelial cells, occasional labeledcells were detected within the lumen of medullary vessels(Figs. 2d, d’). These data show that CoRL proliferation wasnot detected in the cortex, including their original locationin the juxta-glomerulus. However, a subset of CoRL thathad migrated did proliferate when in the medulla.

Renin is not expressed in interstitial CoRLAt 70 days postnatal in a mouse, renin expression isrestricted to the JG [25]. Because our data showed anincrease of interstitial CoRL (i.e. beyond the JG compart-ment) we next determined if these cells retained reninprotein. Double staining for renin and the RFP reportershows that in both sham (not shown) and obstructedkidneys, renin staining was restricted to cells in the JG

only (Additional file 2: Figure S2A and B). These resultssupport the concept that renin lineage cells, when chal-lenged, can take on different cell phenotype and in doingso, no longer express renin [15, 26].

CoRL co-express pericyte markers following UUOBoth CoRL and pericytes derive from Foxd1 lineage cells[10]. CoRL are primarily located in the JG compartmentand afferent arterioles, whereas renal pericytes are typic-ally located around peritubular capillaries and vasa recta[27, 28]. Foxd1 lineage tracking demonstrated that peri-cytes and perivascular fibroblasts are major cellular con-tributors to kidney fibrosis [7, 8]. To determine if CoRLcontribute to pericyte recruitment following a compro-mised microvasculature post UUO, triple staining wasperformed for the reporter (RFP), pericyte markers(PDGFßR, NG2).Staining for endogenous pericyte markers was overall in-

creased in UUO, similar to published data regarding peri-cytes accumulation in fibrosis [7, 8, 29]. In the shamkidney, NG2+/PDGFRß+ staining was detected in mesangialcells, afferent arterioles, peritubular capillaries, and vasarecta (Fig. 3a, d). At d3 post UUO there was an increase ofinterstitial PDGFRß expression, whereas NG2 staining wasfainter in both cortex and medulla (not shown). However,at d7 post UUO, there was an increase of staining for bothPDGFßR and NG2 (Fig. 3b, e), which persisted at d14

Fig. 2 Proliferative and migratory phenotype of CoRL in UUO. (a–c) To detect proliferation in CoRL, double-staining was performed for Red fluorescentprotein (red) to identify CoRL, and BrdU (green) to examine cell proliferation. Nuclei are labeled with DAPI (blue). Medulla: (a) CoRL did not co-expressBrdU in sham kidneys. ( b) At day 7 post UUO, a subset of CoRL co-expressed BrdU (yellow color; arrow shows example). The inset demonstrates this athigher magnification. As expected, other peritubular cells stained for BrdU (open arrowheads). (c) Some dual CoRL+/BrdU+ cells displayed elongatedshape with long cellular processes suggesting cell motility when viewed at high power (inset). (d) To better define the subset of labeledCoRL that appeared to be migrating based on their shape (identified by RFP staining, arrowheads), double staining was performed forthe endothelial cell marker CD31 (green, arrows). (d’) On occasion, CoRL were detected within vessels in the medulla

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(Fig. 3c, f). Triple staining revealed that labeled CoRL(identified by RFP staining) in the interstitium co-expressedboth PDGFßR and NG2 at days 3, 7 and 14 post UUO.The aforementioned results show that labeled CoRL alwaysco-express pericyte markers and this is consistent withthem likely being truly pericytes. In such a capacity, CoRLmay indeed participate in vessel remodeling in UUO.

A subset of pericytes that have detached from vesselsexpress increased αSMATo determine if CoRL become myofibroblasts andlocalize to scarred areas, triple staining was performedfor RFP (CoRL reporter), αSMA (a marker of VSMCwhich appears de novo in myofibroblasts), and CD31(endothelial marker).

Cortical changesIn the sham kidney cortex staining for αSMA and CoRL ispresent in afferent arterioles (Fig. 4a, insets, arrowhead).Post UUO, all interstitial CoRL in the cortex co-expressedαSMA at days 3, 7, 14. At d3, CoRL displayed perivascularlocation (not shown). In addition to peri-endothelial loca-tion (Fig. 4b, insets, arrowhead), CoRL were also foundoutside capillary bed at d7 post UUO (Fig. 4b, arrow). 2w

post UUO when CoRL had migrated away from the ves-sels, they continued to express αSMA, suggesting a myofi-broblast nature (Fig. 4c, insets, arrowhead).

Medullary changesIn the sham kidney medulla, CoRL are present in vasa rectaonly, and these cells co-express αSMA (Fig. 4d, insets,arrowhead). Following UUO, interstitial CoRL co-expressαSMA on days 3, 7 and 14. At d7 CoRL were detectedoccasionally in extravascular locations (not shown), whilealso being peri-vascular (Fig. 4e, insets, arrowheads). By co-staining with CD31 we confirmed that the majority ofCoRL (almost 100 %) that had detached from the under-lying vessels on d14 are αSMA-expressing myofibroblasts(Fig. 4f, insets, arrowheads).

HIF-2α is activated in CoRL in the JG compartment and inafferent arterioles in UUOHypoxia secondary to vasoconstriction and microvascularrarefaction is an important mechanism mediating tubu-lointerstitial injury [30]. In the kidney, HIF-2α is activatedby hypoxia in interstitial fibroblasts-producing erythropoi-etin [31] and in endothelial cells [32, 33]. HIF stabilizationand permanent hypoxic state can be achieved in a mouse

Fig. 3 Interstitial CoRL co-express pericytes markers. To label pericytes, antibody staining against PDGFRß (green) and NG2 (red) was performed,CoRL were identified by RFP staining, nuclei are labeled with DAPI. (a–c) Cortex: (a) Representative images show that in sham-operated mice,CoRL co-express pericyte markers in JG (arrowhead). Insets show high power images of juxtaglomerular CoRL. In UUO, there was an accumulationof interstitial pericytes (orange color). Interstitial CoRL were found in peritubular areas co-expressing pericyte markers at (b) 7 day post UUO,and (c) 14 days post UUO. Insets show high power images of CoRL. (d–f) Medulla: (d) Sham medulla shows CoRL co-expressing pericytemarkers in vasa recta (arrowheads). Insets show high power images. Pericyte marker staining increases in UUO (orange color). Interstitial CoRL were co-labeled with pericyte markers at (e) day 7 post UUO, (f) day 14 post UUO. Insets show high power images of interstitial CoRL

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model by Vhl deletion. Targeted deletion of Vhl in the cellsof renin lineage demonstrates HIF-2α expression along theafferent arterioles, glomerular vascular poles, and intraglo-merular cells [15]. Therefore, cells in the juxta-glomerulusand afferent arterioles respond to hypoxia by activatingHIF-2α. To examine changes in HIF-2α might in CoRLsfollowing UUO, co-staining was performed for RFP (CoRLlabel) and HIF-2α.

Cortical changesIn sham-operated kidneys, HIF-2α staining was detectedin the cytoplasm of a subset of labeled CoRL in the JGcompartment (Fig. 5a, inset, arrowheads). HIF-2α stainingin the cytoplasm is considered the non-active form,whereas when active, HIF-2α translocates to the nucleus

[34]. Following UUO on days 3, 7 and 14, HIF-2α stainingwas detected in the nucleus (co-localized with DAPI) inlabeled CoRL in the JG compartment and afferent arteri-oles (Fig. 5b–d, insets, arrowheads). These subcellularchanges in the location of HIF-2α staining were consistentwith the presence of the inactive form in sham kidneys,but the active form in labeled CoRL post UUO in the JGcompartment.

Medullary changesIn the sham kidney medulla, HIF-2a was not detected inlabeled CoRL (Additional file 3: Figure S3A) In contrastto the cortex, HIF-2α never localized to labeled CoRL inthe medulla post obstruction. Thus was not a falsenegative result because following UUO there was a

Fig. 4 αSMA is expressed by interstitial CoRL in fibrosis. Red fluorescent protein (red) staining identified CoRL, αSMA (green) staining was used to markvascular smooth muscle cells and myofibroblasts, CD31 staining denotes endothelial cells (magenta). Nuclei are labeled with DAPI (blue). (a) In the shamcortex, CoRL were found in JG compartment (arrowheads, insets). (b) At d7 post UUO, CoRL were localized to perivascular area (arrowhead, insets),however, some CoRL were no longer supporting microvessels (arrow). (c) At 14 days post UUO, majority of CoRL (arrowhead) were found outside vascularbed and expressing αSMA suggesting the myofibroblastic conversion. Insets show high power images of merged and single stained panels (d) In thesham medulla, CoRL were found in vasa recta (arrowhead) and co-expressing αSMA. (e) 7 days following UUO, CoRL were still found surroundingendothelial cells (arrowhead, insets). (f) 14 days post UUO, CoRL and localized to avascular areas and expressing αSMA (arrowhead). Insets show highpower images of merged and single stained panels

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progressive increase in HIF-2α staining of interstitialcells of non-CoRL type (Additional file 3: Figure S3B, C,and D, arrowheads).

DiscussionUUO is characterized by hypoxia, interstitial vascular lossand progressive kidney fibrosis. The compensatory mech-anisms that might attempt to counter these events are notwell understood. We demonstrated that following UUO,the number of medullary cells of renin lineage (CoRL)number increases due to cell proliferation and migration.Although CoRL initially appear to be reparative of intersti-tial microvessels following UUO by transdifferentiating into pericyte-like cells, they are likely ultimately injurious bytransdifferentiating into myofibroblast-like cells. Thesechanges are preceded by activation of HIF2a in CoRL inthe juxta-glomerular compartment following UUO.The first major finding from these studies was that the

number of CoRL increases following UUO. We used indu-cible Ren1cCreER xRs-tdTomato-R reporter mice to fatemap a subset of CoRL that were permanently labeled spe-cifically during the period of tamoxifen induction. The dis-tribution of cells-expressing renin is very different duringdevelopment compared to adults [9]. Therefore, labelingmice at 7–8 weeks of age focuses on adult CoRL thatderive from the juxtaglomerular (JG) compartment.

We next asked how might CoRL number increase fol-lowing UUO? A first consideration was migration, fromtheir original location in the JG to the medulla. Severallines of evidence help support this. First, using an indu-cible reporter system, labeled CoRL in the cortex wererestricted to the JG. Thus, the presence of labeled CoRLin the intracapillary loops of some glomeruli, and in thecortical interstitium is in keeping with them migrating.Second, many CoRL in the cortex and medulla had anelongated shape, reminiscent of a migrating cell. Third,several interstitial vessels contained labeled CoRL withintheir lumens. Proliferation is another mechanism thatmight explain the twofold increase in medullary CoRLpost UUO. To this end, we frequently performed BrdUpulse chases to maximally capture cell proliferation.BrdU staining was detected in a subset of CoRL in thecortex and medulla, but not in the JG. Moreover, severalof CoRL with elongated shapes stained for BrdU. Thesedata suggest that once CoRL had moved from the JG, asubset proliferated. However, we cannot exclude thepossibility that native CoRL in the medulla did notproliferate, not migrate. Taken together, the increase inmedullary CoRL post UUO is likely due to a combin-ation of proliferation in CoRL that had migrated awayfrom the JG, migration of non-proliferating CoRL andproliferation of pre-exisiting CoRLs in the medulla.

Fig. 5 Nuclear translocation of HIF-2α in JG and afferent arteriole in UUO. Red fluorescent protein (red) staining identified CoRL, HIF-2α (green)staining was used to detect hypoxia-activated cells. Nuclei are labeled with DAPI (blue). In the sham kidney cortex, there was (a) a cytoplasmicHIF-2α staining in JG cells (inset shows single panel colors, arrowheads indicates the same cell). In UUO kidney, there was an accumulation ofHIF-2α staining in the nuclei in afferent arterioles and JG (insets show single panel colors, arrowheads indicates the same cell) on (b) day 3, (c) day 7,(d) day 14 following kidney injury

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The second major finding in these studies was thetransdifferentiation of medullary CoRL in to pericytes ormyofibroblasts. Several lines of evidence show that CoRLhave marked plasticity under certain conditions [13–16].Accordingly, we next asked what, if any, cell type didCoRL transdifferentiate into in the medulla followingUUO. The first clue that they likely were transdifferen-tiating was the loss of their endocrine function by virtuethat they no longer expressed the renin protein. Second,because at d7 labeled CoRL were largely confined tosurrounding interstitial vessels, we explored the possibil-ity that a subset were transdifferentiating in to pericytes.Indeed, non-renin expressing labeled CoRL co-expressedthe pericyte markers NG2 and PDGFRß. Indeed, CoRLhave been reported to transdifferentiate into pericytesduring development [9] and in glomerular disease [16].We can only speculate that the transdifferentiation of asubset of CoRL to pericytes early in disease is an attemptto maintain or even replace native pericytes, and thusthe interstitial vasculature.Detached pericytes can differentiate into collagen-

producing myofibroblasts [7, 19]. Here, we report thata subset of CoRL later underwent further changes tothat more consistent with a myofibroblast. At day 14post UUO, the majority of labeled interstitial CoRLwere away from any blood vessels. Because theseareas were typified by interstitial fibrosis, we asked ifCoRL were transdifferentiating into myofibroblasts.Indeed, a subset of CoRL did begin to express aSMA,suggesting that they acquired a pro-fibrotic pheno-type. It is not clear which of the following scenariosoccurred first: a decrease in interstitial vessels (i.e. re-duced CD31 staining) forced pericyte-like CoRL todetach, and/or if the detachment of pericyte-likeCoRL lead to unhealthy underlying vessels, followedby rarefaction. Regardless, it is likely that similar toother native cells in the interstitium that acquiremyofibroblast-like features, the subset of CoRL doingso also likely contribute to the increased fibrosis laterin UUO. Finally, our data are in agreement with thecurrent view that Foxd1 lineage cells contribute to fi-brosis [35] especially that CoRL have been recentlyshown to derive from Foxd1+ cells [10].Hypoxia is a common pathway for chronic kidney dis-

ease, including UUO [36, 37]. During kidney obstruction,renal blood flow is reduced as a consequence of pre-glomerular vessel constriction that in turn impairs post-glomerular/peritubular perfusion [38]. HIF-2α is activatedin interstitial and endothelial cells in hypoxia [32, 33].Extracellular matrix deposition further propagates hypoxiato the tubulointerstitium since it increases the distance be-tween capillaries and tubules, decreasing oxygen diffusion[39]. In general, HIF-1 and -2 are both activated in hyp-oxia, but in a different manner: HIF-2 is activated in mild

hypoxia <5 % O2 for many hours (still upregulated after72 h), whereas, HIF-1 is rapidly induced at 1 % O2 to medi-ate acute responses, and declines to the low levels within72 h [40]. This suggests that HIF-2α has a role in the adap-tation to chronic hypoxia, where it is the main regulator oferythropoietin production [41], vascular tumorigenesis[42], cell proliferation [43], and vessel remodeling in dis-ease [44, 45]. Hypoxia is also associated with stem cellphenotype, pluripotent stem cell culture at hypoxic condi-tions (5 % O2) stabilize HIF-2α, increase proliferation andstem cell marker expression [46].The third major finding was that HIF-2α, a hypoxia-

activated factor, is induced in CoRL in the JG and affer-ent arterioles following UUO. The HIF-2α staining pat-tern shifting from a cytoplasmic subcellular locationbefore UUO to a nuclear location HIF-2α on day 3 postUUO is very suggestive of HIF-2α changing from itsinactive form to an active form. Although thisphenomenon preceded the increase in CoRL number inthe intersitium, activated HIF2a persisted in a subset ofCoRL at all time points studied. There is precedence forHIF in CoRL. Kurtz et al. mimicked chronic hypoxia inCoRL by the deletion of Hippel-Lindau protein, andshowed increased HIF-2α expression along the afferentarterioles, glomerular vascular poles, and intraglomeru-lar cells [15]. Also perhaps relevant to the current stud-ies is that the chronic activation of HIF-2α transforms asubset of cells in the JG compartment into fibroblasts-like cells [47]. Taken together, we propose that a likelymechanism that underlies the transdifferentiation ofCoRL in the JG following UUO is the activation of HIF-2α. Further studies are needed to prove if HIF-2α favorsa pericyte-like and/or myofibroblast-like transdifferentia-tion of CoRL in UUO.This study has some limitations. First, the study is

largely descriptive as we focus on association betweenHIF-2α and CoRL activation. However there is sufficientliterature to support our proposed claim that HIF-2α maybe a causative factor leading to CoRL migration and prolif-eration. JG area has been shown to display marked plasti-city in disease settings [13–16]. Also, developmentalstudies demonstrate that arterioles are the source of peri-cyte recruitment [48]. Second, UUO model does notprovide functional data [49, 50]. One may argue thatonly a small number of CoRL is involved in vascularremodelling in UUO, however in an inducible fate-mapping approach only a fraction of cells is labelled (aswe observe glomeruli without labelled CoRL). Import-antly, the strength of this approach is that it allows tofaithfully track the subpopulation of CoRL that werepermanently labeled during tamoxifen induction. Fi-nally, it adds to the pool of known pro-fibrotic progeni-tors arteriolar-derived myofibroblasts. Third limitationof the study is a gender bias since we have only used

Stefanska et al. BMC Nephrology (2016) 17:5 Page 9 of 11

female mice. Most of the studies use male mice andrats and the pro-injury effect of androgens in renal in-jury in males is well documented [51, 52]. Nevertheless,in our UUO model females demonstrated the classicalfeatures of renal injury.

ConclusionsIn summary, this study demonstrated that CoRL areresident kidney progenitors that can respond to vascularinjury by proliferation and migration to possibly partici-pate in vessel remodeling. However, with time, CoRLultimately undergo transition into myofibroblast-likecells, which might favor fibrosis rather than repair. Therole of HIF-2a in CoRL needs further exploration.

Additional files

Additional file 1: Figure S1. UUO induces kidney fibrosis and reducesvascular density in Ren1cCre mice. (A) Following ureteral obstruction ofthe right kidney, the ureter is filled with urine. Non-obstructed kidney(contralateral) undergoes hypertrophy. (B) Reduced weight ratio ofobstructed/non-obstructed kidney on day 7 and 14 after UUO. (C) Endothelialcells, identified by CD31 staining (green), decrease in the cortex and medullafollowing UUO on day 7 and 14 compared to sham kidneys. Examplesof capillary rarefaction are marked with an asterisk (*). (D) PicrosiriusRed staining was examined by polarized light microscopy. Collagenfibers were barely detectable in sham kidneys. Interstitial PicrosiriusRed staining was increased on days 3, 7 and 14 following UUO. (E) Fibrosisquantification based on Picrosirius Red staining score shows a gradual increaseof fibrosis in UUO. Data are represented as mean ± SEM. (PNG 652 kb)

Additional file 2: Figure S2. Renin staining is limited to labeled CoRLin JG and afferent arterioles. Red fluorescent protein staining identifiedCoRL (red), renin staining indicates renin-producing cells (green). DAPI(blue) staining labels nuclei. At 7 days post UUO, (A) in the cortex, reninstaining was detected in classical JG location (arrow) (B) in the medullathere was no renin staining found, interstitial CoRL were negative forrenin. (PNG 431 kb)

Additional file 3: Figure S3. HIF-2α is not activated in interstitial CoRL.Red fluorescent protein (red) staining identified CoRL, HIF-2α (green) stainingwas used to detect hypoxia-activated cells. Nuclei are labeled with DAPI (blue).(A) Sporadic HIF-2α-expressing cells were found in sham kidney. FollowingUUO, there was an increase of HIF-2α-activated cells (arrowheads) at (B) d3,(C) d7, (D) and d14. However, none of interstitial CoRL expressed HIF-2α(arrows). (PNG 1063 kb)

AbbreviationsCoRL: cells of renin lineage; HIF-2α: hypoxia inducible factor-2α;JG: juxtaglomerular; PDGFß-R: platelet derived growth factor Beta receptor;RFP: red fluorescent protein; TR: tomato red; UUO: unilateral ureteral obstruction.

Competing interestsThe authors declare that they have no competing interests.

Authors’ contributionsSS, JP, AS: study design, AS: collection and analysis of data, and drafting of themanuscript, SS, KG, JD &JP: critical revision of the article for important intellectualcontent and final approval of the article. DE&NK: critical roles in immunostainingand generating transgenic mice. All authors read and approve the finalmanuscript.

AcknowledgmentsThis work was supported by NIH grants R24 DK094768-01 and R01DK093493-02 to J.S.D. and S.J.S.

Author details1Department of Medicine, Division of Nephrology, University of Washington,Seattle, WA 98104, USA. 2Department of Molecular and Cellular Biology,Roswell Park Cancer Institute, Elm and Carlton Streets, Buffalo, NY 14263,USA. 3Biogen Idec, Cambridge, MA 02142, USA.

Received: 31 August 2015 Accepted: 22 December 2015

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